Sample collection method

ABSTRACT

The present invention relates to methods for the collection of a sample from faecal material and, further, the detection of occult blood in or on the faecal material via the testing of the sample collected from the faecal material. The present invention also relates to collection methods comprising the use of a brush-like device having flexible or semi-flexible bristles wherein the brush-like device is contacted to the faecal material. The present invention also relates to detection of occult blood from the sample collected from the faecal material by means of a guaiac test or immunochromatographic test. The present invention additionally relates to the detection of one or more indicators of a pathological condition in or on the faecal material from which the sample is derived.

FIELD OF THE INVENTION

This invention relates to a method for collecting a sample forsubsequent use in the detection of an analyte in the sample. In oneparticular embodiment, this invention relates to a method for samplingfaecal material for the purposes of subsequent detection in the sampleof occult blood or one or more other indicators of a pathologicalcondition.

The present invention also extends to an assay kit which is particularlysuitable for the purposes of detection in a sample derived from faecalmaterial of occult blood or one or more other indicators of apathological condition.

BACKGROUND OF THE INVENTION

A well known and widely-used clinical reagent for the detection ofoccult blood in a sample, particularly a faecal sample, is guaiac (alsoknown as gum guaiac or resin guaiac). When used in association with anappropriate developer solution, guaiac provides a colorimetric assaysystem for detecting haemoglobin in the sample. Such tests arecommercially available, for example, Hemoccult II and Hemoccult II Sensa(SmithKline Diagnostics, San Jose, Calif., USA).

Prior Australian Patent No. 665956 (International Patent Application No.PCT/US92/04425) notes that among the many analytical systems used fordetection and/or determination of analytes, particularly analytes ofbiological interest, are chromatographic assay systems. Among theanalytes of biological interest frequently assayed with such systemsare:

-   -   1. hormones, such as human chorionic gonadotropin (hCG),        frequently assayed as a marker of human pregnancy;    -   2. antigens, particularly antigens specific to bacterial, viral,        and protozoan pathogens, such as Streptococcus, hepatitis virus,        and Giardia;    -   3. antibodies, particularly antibodies induced as a result of        infection with pathogens, such as antibody to the bacterium        Helicobacter pylori and to human immunodeficiency virus (HIV);    -   4. other proteins, such as haemoglobin, frequently assayed in        determinations of faecal occult blood, an early indicator of        gastrointestinal disorders such as colon cancer;    -   5. enzymes, such as aspartate aminotransferase, lactate        dehydrogenase, alkaline phosphatase, and glutamate        dehydrogenase, frequently assayed as indicators of physiological        function and tissue damage;    -   6. drugs, both therapeutic drugs, such as antibiotics,        tranquillisers and anticonvulsants, and illegal drugs of abuse,        such as cocaine, heroin, and marijuana; and    -   7. vitamins.

Such chromatographic systems are frequently used by physicians andmedical technicians for rapid in-office diagnosis and therapeuticmonitoring of a variety of conditions and disorders. They are alsoincreasingly used by patients themselves for at-home monitoring of suchconditions and disorders.

Among the most important of such chromatographic systems are the “thinlayer” membrane-based systems in which a solvent moves as a solventfront across a thin, flat absorbent medium (e.g., nitrocellulosemembrane). Among the most important of tests that can be performed withsuch thin layer systems are immunoassays, which depend on the specificinteraction between an antigen or hapten and a corresponding antibody.The use of immunoassays as a means of testing for the presence and/oramount of clinically important molecules has been known for some time.

Chromatographic techniques used in conjunction with immunoassays includea procedure known as immunochromatography. In general, this techniqueuses a disclosing reagent or particle that has been linked to anantibody to the analyte to be assayed, forming a conjugate. Thisconjugate is then mixed with a specimen and, if the analyte to beassayed is present in the specimen, the disclosing reagent-linkedantibodies bind to the analyte to be assayed, thereby giving anindication that the analyte to be assayed is present. The disclosingreagent or particle can be identifiable by colour, magnetic properties,radioactivity, specific reactivity with another molecule, or anotherphysical or chemical property. The specific reactions that are employedvary with the nature of the analyte being assayed and the sample to betested.

The present invention is particularly, but not exclusively, directed tocollection of samples derived from faecal material for occult blooddetection, for example in screening for colorectal cancer. As previouslydescribed, guaiac testing provides a colorimetric assay system fordetection of haemoglobin in a sample, however because of the largenumber of false positives obtained in guaiac testing, in screeningprograms the use of two or three guaiac tests has been recommended,confirmed when positive by an immunological test for human haemoglobin(Favennic L., Kapel N., Meillet D., Chochillon C. and Gobert J. G.,Annales de Biologie Clinique, 50(5):311-3, 1992). More recently, acombination of guaiac and immunological testing has been suggested(Allison, J. E., Tekawa, I. S., Ransom, L. J. and Adrian, L. L. N. Engl.J. Med., 334:155-9, 1996).

It is an object of the present invention to provide a sample collectionmethod which is simple and economic, and which enables subsequentdetection and/or determination of analyte in the sample to be readilycarried out, for example using a guaiac test, and/or animmunochromatographic or other immunodiagnostic procedure.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method forcollecting a sample derived from faecal material, comprising contactingthe faecal material with a fluid and subsequently collecting a sample ofthe fluid with a brush or brush-like device having flexible orsemi-flexible bristles, wherein the sample of the fluid is collectedwithin the bristles of the brush or brush-like device.

Preferably, the fluid is water.

The term “brush” as used herein is used to denote device comprising astem or handle, usually elongate, and a clump, bunch or group ofbristles, hair or other similar flexible or semi-flexible elongatestrands, laminar flaps or the like attached to the stem or handle. Theterm “brush-like device” is used herein to denote a device which issimilar to a brush in that it includes a bunch, clump or group ofbristles, hair or other similar flexible or semi-flexible elongatestrands, laminar flaps or the like. Whilst reference is made throughoutthe present specification to the collection of a sample within thebristles of a brush or brush-like device, it is to be understood thatthe reference to “bristles” is used to include the hairs or othersimilar flexible or semi-flexible elongate strands, laminar flaps or thelike of a brush or brush-like device.

Preferably, the bristles of the brush or brush-like device will have alength of about 0.2 to 3 cm long, more preferably a length of 1 to 2 cm.

In another embodiment, the present invention also extends to an assaykit for testing faecal material which comprises a sample collectiondevice which is a brush or brush-like device having flexible orsemi-flexible bristles, together with means for detection of an analytein a sample derived from faecal material.

Such an assay kit is particularly suited for use in detection of occultblood in a sample derived from faecal material. The detection of occultgastrointestinal bleeding is a common method for screening forcolorectal cancer. Commonly referred to as the aecal occult blood (FOB)test, a variety of formats are known in the art (see, for example, U.S.Pat. Nos. 3,996,006; 4,225,557; 4,789,629; 5,064,766; 5,100,619;5,106,582; 5,171,528; 5,171,529; and 5,182,191). The majority of testformats are based on the chemical detection of the heme groups presentin faecal material as a breakdown product of blood. In such tests, thepseudoperoxidase nature of the heme group is used to catalyse acolorimetric reaction between an indicator dye and peroxide. The oxygensensitive dye can be gum guaiac, orthodianisidine, tetramethylbenzidine,or the like, with guaiac being preferred.

The means for detection of an analyte in a sample which is incorporatedinto an assay kit as described above may, for example, be means forcarrying out a guaiac test for the detection of occult blood in thesample. Alternatively, or additionally, the means for detection of ananalyte in a sample may be means for detection of occult blood (or otherdiagnostic antigens) in the sample by means of a chromatographicprocedure, particularly by an immunochromatographic or otherimmunodiagnostic procedure which is well known in the art. Suitableimmunochromatographic procedures are described, by way of example, inU.S. Pat. Nos. 5,591,645 and 5,622,871, the disclosures of which areincorporated herein by reference.

Whilst the present invention is particularly useful in FOB testing asdescribed in detail herein, it is to be understood that the method andassay kit as broadly described herein may be used in sampling faecalmaterial and subsequent testing of the sample to detect the presence ofone or more other indicators of a pathological condition, for example,tumour-derived antigens, in addition to or instead of FOB testing.

Throughout this specification, unless the context requires otherwise,the word “comprise”, and or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

DETAILED DESCRIPTION OF THE INVENTION

In the most preferred embodiment, the present invention relates to theuse of a brush as a device for obtaining a sample derived from faecalmaterial, and particularly stool, in a fluid such as water, particularlyfor the detection of occult blood as an indicator of colorectal cancer(CRC) or its precursor conditions.

Most existing faecal occult blood tests (FOBTs) use a sampling stick orpaddle to take smears directly from the surface of a collected faecalsample. European Patent Application No. EP 0 727653 discloses the use ofa brush device having stiff bristles to collect a sample from thesurface of faecal material directly on the bristles. Many CRCs or theirprecursors (e.g. adenomas>1 cm), bleed into the lumen of the smallintestine. As these malignancies arise as protrusions from the wall ofthe intestine they make contact with the surface of the stool in theirregion of contact as the stool passes that point. The blood, therefore,may not be evenly distributed through or over the stool. As a result,existing tests that rely on surface sampling of the stool may or may notsample from that portion of the stool where blood is present.

If the stool or other faecal material is sampled in a fluid, forexample, when it is in the water of the toilet bowl, there is a betteropportunity to gain a representative sampling of the whole stool. Thisis particularly the case where a small brush (e.g. a small artist'spaint brush having bristles about 1 to 2 cm in length) is used forsampling. A brush may be used to “paint” the surface of the stool so asto displace any blood on the surface of the stool into the watersurrounding the stool. The flexible or semi-flexible bristles of thebrush will be relatively “open” during this brushing and samplingprocess, but will “close” as the brush is withdrawn from the water,thereby keeping a ample of the water (and any blood contained therein),surrounding the stool within the interstitial spaces of the bristles.This sample may then be transferred to a suitable assay device forsubsequent testing.

By way of contrast, if an absorbent sampling device, such as a swab, wasused for sampling, water would infiltrate the fibre windings of the swabon its first contact with the water in the toilet bowl. In this case,there would be little chance of effective displacement of theinfiltrated water by any blood-containing water in the vicinity of thestool, and as a result the sampling procedure would not effectivelysample any such blood-containing water.

Alternatively, if a solid sampling device such as a solid sampling stickor paddle, or a loop or barbed probe was used, the water sampled fromaround the stool would be lost as the device was withdrawn through thewater of the bowl, and once again the sampling procedure would noteffectively sample any blood-containing water.

A further advantage which is obtained by the use of a brush orbrush-like sampling device in accordance with the present invention isthat the fluid sample collected within the bristles of the samplingdevice as described above is collected in a semi-quantitative manner, inthat the amount of fluid held within the interstitial spaces of thebristles of the sampling device will be a reasonably constant amount forany particular size and configuration of the sampling device.

As described above, an important feature of the sampling device is thatthe bristles of the device, as defined above, are flexible orsemi-flexible. This enables the device to be used to obtain a sample offluid surrounding the faecal material into which any occult blood on orat the surface of the faecal material has been dispersed, instead ofattempting to obtain a sample directly from the surface of the faecalmaterial where it may only be present in isolated locations, andaccordingly where there is a risk that any sample taken directly fromthe surface of the faecal material may not be taken from a locationwhere any blood is present.

As previously described, colorectal cancers and adenomas often bleedinto the lumen of the large bowel. Initially, only a small, localisedamount of blood leakage may occur, leading to isolated spots or areas ofblood occurring on the surface of faecal material in the large bowelwhich will be exposed to the blood first. It is not unreasonable toassume that much of this blood will remain on the surface of this faecalmaterial after it is passed. Similarly, almost all colorectal cancersand all adenomas occupy only a small portion of the diameter of thelarge bowel. Therefore, it is also likely that the blood from suchlesions will be striped along the faecal material. If this is the case,the brush method of the present invention for sampling faecal materialwill have an advantage over traditional FOBT sampling methods becausethe sampling method of the present invention takes a more representativesample than that of the traditional methods.

Further features of the present invention are more fully described inthe following Example(s). It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLE 1

The suitability of a brush for sampling blood in water has been shown tobe effective by several means:

-   1. Blood (10 μL) was added to water (50 mL) in a beaker. After the    blood settled to a discrete drop at the bottom of the beaker, a    brush (#5, LiFung, Hong Kong) was first used to sample the surface    water from the beaker. This sample tested negative in a faecal    occult blood (FOB) test (Enterix). After mixing the contents of the    beaker, a second, similar brush was shown to be capable of    selectively sampling sufficient of the blood to be detected in a    similar FOB test.-   2. A stool sample was injected with blood (50 μL) so that the blood    was sequestered within a crevice in the stool. The stool was added    to a toilet bowl and brushes as described above were used to sample:    -   (a) The water of the bowl.    -   (b) The water surrounding the stool after the surface of the        stool was “brushed”.        When tested in FOB tests (Enterix), samples (a) tested negative        for blood, whereas samples (b) tested positive. In this        experiment it may be expected that the sequestered blood would        have been missed by conventional sampling of the stool surface        with a stick or paddle.

3. Table 1 below shows the results of a series of experiments to testthe effectiveness of sampling of stool samples with a brush as describedabove. Blood was added directly to normal stool samples, before or afterthe deposition of the stools into a toilet bowl. Normal stools and thebowl water before stool addition were also sampled. In each case samplescollected by the brush method were tested for the presence of blood byan FOB test (Enterix). TABLE 1 Normal stool 25 μL 50 μL 100 μL FOB Test(i.e. no blood blood blood Results Bowl Water addition) added addedadded No. positive — — 4/4 15/15 27/27 No. negative 2/2 15/15 — — —

As shown in the Table, all toilet bowl water and normal stool samplestested negative in the FOB test, whereas all samples with added blood(≧25 μL) gave a positive test result. These results compare favourablywith the sensitivity and specificity data reported with tests that usedirect stool sampling with a sampling stick Rosen, P., Knaai, J. andSamuel, Z. Dig. Dis. Sci., 42(10):2064-71, 1997).

EXAMPLE 2

The aim of this study was to determine if the sampling method of thepresent invention is more capable of detecting significant quantities ofblood than a traditional method of FOBT sampling when the blood isstriped along one side of the surface of a stool.

Methods

Ten faecal samples were collected from three individuals and spiked withblood to a concentration of 0.5 milligrams of haemoglobin per gramfaeces. Spiking was achieved by spotting the blood along the surface ofthe stool in a stripe.

Five spiked stools were tested both by the method of the presentinvention (EnterixOBT) and by FlexSureOBT (Beckman Coulter Personal CareDiagnostics, Palo Alto, Calif., U.S.A.). The samples for testing werecollected as per the manufacturer's instructions for each test exactlyas if the person had been defecating directly into the toilet bowl(EnterixOBT) or into a paper saddle (FlexSureOBT). In the EnterixOBTtest, the sampling device is a brush (LiFung, Hong Kong) having aplastic stem or handle (approx. 185 mm length, 4-6 mm diameter) andflexible bristles (approx. 15 mm length). The sampling device for theFlexSureOBT test is a solid paddle or “popsicle” stick. To avoid bias,sampling for each test was standardised. and blinded For EnterixOBT,samples were collected by five brush strokes of the upright surface ofthe stool. Where loose stools were concerned, the brush was swirledaround the stool five times. For FlexSureOBT, sampling was carried outas per manufacturer's instructions at random points on the stool.

All tests were developed three-four days after sampling and all testswere read by two independent readers. The results are shown in Table 2below.

Results TABLE 2 Test results for stripe-spiked stool samples. EnterixOBTFlexSureOBT (n = 5) (n = 5) Reader A Reader B Reader A Reader B Positive5 5 1 1 Negative 0 0 4 4Discussion

Although the number of samples tested in this study is small, EnterixOBTappears to be able to detect a significant quantity of blood better thanFlexSureOBT when the blood is striped along the surface of the stool.This difference is presumably due to the different methods of samplingemployed by each test. As a result, EnterixOBT appears to have a clearadvantage over FlexSureOBT in terms of the clinical detection of occultblood on faecal material, for example, in the detection of colorectalneoplasia.

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

1. A method for collecting a sample derived from faecal material,comprising (i) contacting the faecal material with a fluid to disperseany blood present in or on the faecal material into the fluid, and (ii)subsequently collecting a sample of the fluid.
 2. A method according toclaim 1, wherein the fluid is water.
 3. A method according to claim 1 orclaim 2, wherein said contacting step comprises physically contactingthe surface of the faecal material to displace any blood on the surfaceof the faecal material into the fluid.
 4. A method according to claim 3,wherein the surface of the faecal material is brushed or painted with abrush or brush-like device having flexible or semi-flexible bristles todisplace any blood on the surface of the faecal material into the fluid.5. A method according to claim 1 or claim 2, further comprising the stepof testing the sample to detect occult blood in or on the faecalmaterial from which the sample is derived.
 6. A method according toclaim 5, wherein the presence of occult blood, if any, in or on thesample is detected by means of a guaiac test.
 7. A method according toclaim 5, wherein the presence of occult blood, if any, in or on thesample is detected by means of an immunochromatographic test.
 8. Amethod according to claim 1 or claim 2, further comprising the step oftesting the sample to detect one or more indicators of a pathologicalcondition in or on the faecal material from which the sample is derived.9. A method according to claim 8, wherein said one or more indicators ofa pathological condition comprise one or more tumour-derived antigens.10. A method for the detection of occult blood in faecal material,comprising (i) contacting the faecal material with a fluid to disperseany blood present in or on the faecal material into the fluid, (ii)subsequently collecting a sample of the fluid, and (iii) detecting thepresence of blood, if any, in the sample.
 11. A method according toclaim 10, wherein the fluid is water.
 12. A method according to claim 10or claim 1 1, wherein said contacting step comprises physicallycontacting the surface of the faecal material to displace any blood onthe surface of the faecal material into the fluid.
 13. A methodaccording to claim 12, wherein the surface of the faecal material isbrushed or painted with a brush or brush-like device having flexible orsemi-flexible bristles to displace any blood on the surface of thefaecal material into the fluid.
 14. A method according to claim 10,wherein the presence of blood, if any, in or on the sample is detectedby means of a guaiac test.
 15. A method according to claim 10, whereinthe presence of blood, if any, in or on the sample is detected by meansof an immunochromatographic test.